PS974 MODELING NKG2D-BASED CAR-T CELL THERAPY IN ANIMALS WITH ACUTE MYELOID LEUKEMIA

Abstract

Background: Pharmacologic inhibition of pro-survival Bcl-2 family members (Bcl-2, Mcl-1, and BCL-xL) has been extensively studied for acute myeloid leukemia (AML) therapy. This has led to the recent approval of the Bcl-2 inhibitor venetoclax. Primary resistance to venetoclax is commonly associated with overexpression of Mcl-1 and Bcl-xL, suggesting heterogeneity in dependence on Bcl-2 family members between AML patients. In healthy bone marrow (BM), Bcl-xL is essential for effective development of the erythroid lineage. Pure erythroid leukemia (PEL) and erythroleukemia (MDS), formerly classified as acute erythroid leukemia (AEL, FAB M6), are malignancies arising from the erythroid lineage. Thus, we hypothesized that targeting BCL-xL could represent a viable therapy option for leukemias with erythroid features. Aims: We aimed to assess the expression levels of Bcl-2 family members across different leukemia subtypes and to study whether Bcl-xL would be a viable drug target for AEL using large-scale functional genomic and ex vivo drug screening data. Methods: Publicly available transcriptomic datasets, including DMAP, TCGA, HEMAP, and CCLE, were used to compare gene expression in healthy samples, primary leukemia samples and AML cell lines. Genome-wide RNAi and CRISPR-Cas9 loss-of-function screening data from the DepMap project was used to evaluate essential genes for AEL cell lines. Drug screening was performed with 528 compounds across a 10,000-fold concentration range using 21 AML cell lines involving five AEL cases (F-36P, OCI-M1, TF-1, HEL, and KG-1). Protein levels of Mcl-1, Bcl-2, and Bcl-xL were assessed from the same cell lines with Western blot analysis. Drug testing was also performed for three primary AEL patient samples. Results: BCL2L1 (Bcl-XL) was strikingly overexpressed in the healthy erythroid lineage (DMAP) and also in the AEL (FAB M6) when compared to other leukemias and AML FAB subtypes in both TCGA and Hemap datasets (Figure). Reflecting the expression pattern in primary patient samples, AML cell lines derived from AEL expressed elevated levels of BCL2L1 and decreased levels of MCL1 and BCL2. Interestingly, also AML cell lines derived from megakaryoblastic leukemia (CMK and M-07e) showed similar trend. The analysis of genome-wide RNAi and CRISPR-Cas9 screening data identified BCL2L1 as the most selective vulnerability in the erythroid and megakaryocytic cell lines (HEL, TF-1, CMK, MOLM16, and F-36P) when compared to other 18 cell lines belonging to different AML FAB subtypes. Accordingly, amongst the 528 anticancer compounds Bcl-xL inhibitors, A-1331852 and A-1155463, were the most selectively effective compounds in erythroid and megakaryocytic cell lines. In contrast, venetoclax targeting Bcl-2 and S-63845 targeting Mcl-1 were effective in other AML subtypes but not in AEL cell lines. The observed druggable antiapoptotic protein dependencies were also reflected in the protein levels. Finally, primary AEL samples showed pronounced sensitivity to A-1331852 and A-1155463 when compared to other AML samples.Summary/Conclusion: Given the high expression of Bcl-xL in erythroid leukemia and the lack of molecularly targeted therapy options, Bcl-xL is a promising target for further exploration in treatment of leukemias with erythroid features.